New data from diet restricted Lepob (ob/ob) mutant C57BL/6J mice-our DR-OB model-challenge the paradigm that benefits of diet restriction (DR) require insulin sensitivity, low insulin, and low adiposity. DR-OB mice have severe insulin resistance, high insulin levels, as well as high adiposity, but live as long as diet restricted controls (DR +/?) that are insulin sensitive with low insulin and low adiposity. This model will be used to test three categories of mechanisms proposed to produce the beneficial effects of DR: 1) the insulin, IGF-1, mTOR, and nutrient signaling transduction pathways, 2) fuel utilization pathways, 3) adipose tissue function. Five groups of mice will be compared at 6, 12, and 22 months of age: DR-OB, partially restricted ob/ob (partial DR-OB), ad lib fed ob/ob (AL-OB), diet restricted and ad lib fed genetic controls (DR +/?) and (AL +/?). Signal transduction, fuel utilization, and adipose tissue markers that differ between DR-OB and DR +/? will identify candidate mechanisms that correlate with low insulin and low adiposity but do not increase lifespan. Markers that are similar in DR-OB and DR +/?, and that correlate with lifespan in DR-OB, partial DR-OB and AL-OB, will specify candidate mechanisms by which DR may increase lifespan, which will be further tested later. The current project is unique in that it distinguishes features of DR-associated hypoinsulinemia and leanness that extend lifespan from features that do not. This work will specify the set of candidate mechanisms comprising those critical for DR-mediated, extended lifespan by testing the following hypotheses: Aim 1. That, in DR-OB mice, critical branches of the mTOR, AMP-kinase, and insulin/IGF-1 signal transduction pathways shift from patterns typical of insulin resistance, hyperinsulinemia and obesity to patterns typical of DR and extended longevity. Key intermediates of these pathways will be tested in liver and gastrocnemius muscle, using western blots to compare protein pool and site-specific phosphorylation levels. Aim 2. That, in DR-OB mice, fuel utilization pathways shift-at or before a critical point necessary to extend lifespan-from patterns typical of metabolic syndrome to patterns typical of DR that reduce mitochondrial free radical production. In vivo tests to identify this point will include short-term fasting glucose and insulin levels, glucose tolerance, insulin sensitivity, gluconeogenesis, glycogenolysis, beta-oxidation, glucose utilization, lipid utilization, and 24-hr respiratory exchange ratio. Collaborators will test free radical damage to proteins of the electron transport chains for liver and muscle mitochondria. Aim 3. That, in DR-OB mice, critical aspects of adipose tissue reflect the lean state of DR +/? mice rather than the obese state of AL-OB mice. The following will be determined: body composition and subcutaneous/visceral fat tissue distribution (using computerized tomography imaging); circulating adipokines (adiponectin, resistin, IL-6, and TNF-alpha); circulating and tissue levels of free fatty acids, triglyceride and cholesterol; and fat cell size distribution in mesenteric and subcutaneous fat tissue.